Bacterial receptor structures

ABSTRACT

Novel proteins obtainable by mutagenesis of surface-exposed amino acids of domains of natural bacterial receptors, said proteins being obtained without substantial loss of basic structure and stability of said natural bacterial receptors; proteins which have been selected from a protein library embodying a repertoire of said novel proteins; and methods for the manufacture of artificial bacterial receptor structures.

This applications is a 371 of PCT/SE95/00034 filed Jan. 10, 1995 whichis a Foreign/PCT application of 9400088-2 filed Jan. 14, 1994.

The present invention relates to a new bacterial receptor structuresoriginating from natural bacterial receptor structures which have beenmodified in regard to amino acid residues involved in the originalinteraction function, whereby said original interaction function hasbeen substantially inhibited and replaced by a modified interactionfunction directed to a desired interaction partner.

Several bacteria known to invade mammals have evolved surface proteinscapable of binding to a variety of substances including host specificcarbohydrates and proteins. Several such receptors from Gram-positivebacterial pathogens have been isolated and characterized in detail aswill be shown below. Most well-characterized are the Fc receptors, namedafter the capability of binding to the constant Fc part of IgG. Based onbinding experiments to IgG from various mammalian sources, andsubclasses thereof, Fc receptors have been divided into six types I-VI.The receptor from S. aureus, protein A SPA!, defining the type Ireceptor, has been the subject of immense studies.

SPA binds IgG from most mammalian species, including man. Of the foursubclasses of human IgG, SPA binds to IgG1, and IgG4 but shows very weakor no interaction with IgG3 Eliasson, M. et al, 1989 J. Biol. Chem.9:4323-4327!. This pseudoimmune reaction has been used for more than 20years for the purification and detection of antibodies in diagnostic,research and therapeutic applications. Cloning, sequencing andEscherichia coli expression of defined fragments of the SPA generevealed a highly-repetitive organization, with five IgG binding domainsE-D-A-B-C!, a cell wall spanning region and membrane anchoring sequenceXM! Uhlen, M. et al, 1984 J. Biol. Chem. 259:1695-1702; Moks, T. et al,1986 Eur. J. Biochem. 156:637-643!. A vast number of plasmid vectorshave been constructed, allowing gene fusions to different fragments ofthe gene for the purpose of fusion protein production in different hostsNilsson B. and Abrahmsen, L. 1990 Meth. Enz. 185:144-161! (FIG. 2a).

The structure for a complex between human Fc IgG1! and a single domainB! of SPA has been determined by X-ray crystallography at a resolutionof 2.8 Å Deisenhofer, J. et al 1981 Biochemistry 20:2361-2370!. Based onthis structure and additional information from NMR experiments, the Bdomain can be viewed as a compact structure consisting of threeanti-parallel α-helices connected with loops. In the Fc binding, whichis of both electrostatic and hydrophobic nature, only side chains ofresidues from helices 1 and 2 are involved, whilst the third helix isnot participating in the binding. Based on this domain B, a syntheticIgG-binding domain Z! Nilsson, B. et al 1987 Prot. Eng. 1:107-113! hasbeen constructed, suitable as fusion partner for the production ofrecombinant proteins which allows purification by IgG affinitychromatography. The high solubility and the stable structure of the Zdomain has been utilized for production, purification and renaturationof a large number of recombinant proteins. Josephsson, S. and Bishop, R.Trends Biotechnol. 6:218-224; Samuelsson, E. et al 1991 Bio. Technol.9:363-366!.

Streptococcal strains of serological groups C and G display a bindingrepertoire for mammalian IgGs, including human IgG3, which is evenbroader than for the type I receptor. The name protein G was suggestedfor this type III receptor from group G streptococci. In 1986 Olsson andco-workers reported on the cloning and sequencing of the gene from theserological group G streptococci G148! Guss, B. et al, 1987 EMBO J.5:1567-1575; Olsson, A. et al, 1987 Eur. J. Biochem. 168:319-324!. Inanalogy with SPA is SPG a repetitively arranged molecule, comprising anIgG-binding region of three homologous domains C1,C2,C3!, spaced bysmaller D-regions (FIG. 2A). Compared to SPA, SPG displays a differentbinding spectra for immunoglobulins from different species andsubclasses thereof. The IgG binding domains of protein G are now widelyused as an immunological tool, i.e. in the affinity purification ofmonoclonal antibodies. Production of subfragments constructed byDNA-technology, have shown that an individual C-region is sufficient forfull IgG-binding. Recently, the structure for a complex between theC1-domain from SPG and human Fc was determined with X-raycrystallography (FIG. 2B). This shows that SPG binds to the CH2-CH3interface but at a different site compared to SPA. The binding is mainlyof electrostatic nature which is in contrast to the large contributionof hydrophobic forces seen for the SPA-Fc interaction. Moreover, the 3-Dstructure of C1 differs from the X structure in that it is built up bytwo β-sheets connected by an α-helix ββ-α-ββ!. The residues of C1 whichaccording to the structure are involved in the binding, corresponds tothe α-helix, the loop and the following β-sheet.

An additional activity of SPG is the capability to bind serum albumin.The binding strength is species dependent, and among the tested samples,SPG binds strongest to serum albumin from rat, man and mouse. Productionand binding studies of subfragments of SPG shows that the two bindingactivities are structurally separated and that the serum albumin bindingfunction is located at the repetitive A-B region Nygren et al 1990 Eur.J. Biochem. 193:143-148!. This region has been used for severalbiotechnological purposes. Recombinant proteins have been produced asfusions to the region which enables the purification by affinitychromatography, where human serum albumin most frequently has been usedas immobilized ligand. Proteins found to be proteolytically sensitivehave been produced as "dual affinity fusions" in which they are flankedby two different affinity tails derived from SPA and SPG, respectively.Purification schemes employing both the N- and C-terminal are thuspossible which ensures the recovery of an intact target proteinHammarberg et al 1989 Proc. Natl. Acad. Sciences USA 96:4367-4371!. Thestrong and specific binding to serum albumin has also been used for thein vivo stabilization of therapeutic proteins.

Through complex formation with the very long-lived serum albumin, thereceptor is carried in the circulation (macaque apes) with a half-lifewhich is close to the half-life for serum albumin itself. Studies inmice with the for HIV/AIDS therapy interesting, but rapidly clearedT-cell receptor CD4, showed that is was substantially stabilized whenfused to the serum albumin binding region, when compared with an unfusedcontrol protein Nygren et al 1991 Vaccines 91 Cold Spring Harbor Press363-368!. The slow clearance can probably be explained by the complexformation with serum albumin which circumvents elimination by the liverand excretion in the kidney.

In order to determine the minimal extension required for maintainedbinding to serum albumin, several smaller fragments of the A-B regionhave been produced and analyzed. The smallest fragment so far with serumalbumin binding activity is a 46 residue fragment "B2A3"! comprisingregion A3 flanked by 13 and 9 residues, respectively, from regions B2and S.

Based on homology and binding studies of other partial fragments SPG isregarded to be trivalent with regard to binding to serum albumin.Similar to the monovalent IgG-binding domains Z and C1 B2A3 isrelatively small and shows high solubility and stability and istherefore a suitable candidate for modification.

SUMMARY OF THE INVENTION

The present invention has for its main purpose to provide new bacterialreceptor structures by modifying natural bacterial receptors in regardto their original interaction functions to result in new structureshaving modified interaction functions.

Another object of the invention is to provide artificial bacterialreceptor structures which are stable and more resistant to variousconditions, such as increased temperatures.

Yet another object of the invention is to provide artificial bacterialreceptor structures, the interaction functions of which have beenmodified to direct same to other desired interaction partners.

With these and other objects that will be clear from the followingdisclosure in mind the invention provides for novel proteins obtainableby mutagenesis of surface-exposed amino acids of domains of naturalbacterial receptors said proteins being obtained without substantialloss of basic structure and stability of said natural bacterialreceptors. Said proteins have preferably been selected from a proteinlibrary embodying a repertoire of said novel proteins. In such novelbacterial receptor structures, at least one amino acid residue involvedin the interaction function of the original bacterial receptor has beenmade subject to substitution by another amino acid residue so as toresult in substantial loss of the original interaction capacity with amodified interaction capacity being created, said substitution beingmade without substantial loss of basic structure and stability of theoriginal bacterial receptor.

It is preferred that said bacterial structures originate fromGram-positive bacteria. Among such bacteria there may be mentionedStaphylococcus aureus, Streptococcus pyogenes group A!, Streptococcusgroup C,G,L, bovine group G streptococci, Streptococcus zooepidemicusgroupC!, Streptococcus zooepidemicus S212, Streptococcus pyogenes groupA!, streptococci groups A,C,G, Peptostreptococcus magnus, Streptococcusagalactiae group B!.

Of special interest are thermophilic bacteria evolved to persist inenvironments of elevated temperatures. Receptors from species like e.g.Bacillus stearothermophilus, Thermus aquaticus, Thermococcus litoralisand Pyrococcus have the potential of being naturally execptionallystable, thus suitable for providing structural frameworks for proteinengineering according to the invention.

It is particularly preferred to use as a starting material for themodification of the interaction function bacterial receptor structuresoriginating from staphylococcal protein A or streptococcal protein G.

Among preferred receptors there may be mentioned bacterial receptorsoriginating from Fc IgG!receptor type I, type II, type III, type IV,type V and type VI, fibronectin receptor, M protein, plasmin receptor,collagen receptor, fibrinogen receptor or protein L K light chains!,protein H human IgG!, protein B human IgA,A1!, protein Arp human IgA!.

Particularly preferred bacterial receptors originate from the FcIgG!receptor type I of staphylococcal protein A or the serum albuminreceptor of streptococcal protein G.

In order to maintain stability and the properties of the originalbacterial receptor structure it is preferred in accordance with thepresent invention that the substitution involving amino acid residuestaking part in the interaction function of the original bacterialreceptor does not involve more than about 50% of the amino acid residuesof the original bacterial receptor. It is particularly preferred thatnot more than about 25% of the amino acid residues of the originalbacterial receptor are made subject to substitution.

In regard to the original bacterial receptor structures selected formodification of their interaction functions it is particularly preferredto use receptors originating from the IgG-binding domains, Z, C1, andthe serum albumin binding domains B2A3.

In order to maintain as far as possible the stability and properties ofthe original receptor structure subject to modification in accordancewith the present invention it is preferred that the substitution thereofinvolves not more than substantially all of the amino acid residuestaking part in the interaction function of the original bacterialreceptor.

In order to obtain favourable properties concerning stability andresistance to various conditions it is preferred that the bacterialreceptor according to the present invention is comprises of not morethan about 100 amino acid residues. It is known from scientific reportsthat proteins of a relatively small size are fairly resistant toincreased temperatures and also to low pH and certain chemicals. Fordetails concerning temperature resistance c.f. the article by Alexanderet al. in Biochemistry 1992, 31, pp 3597-3603.

With regard to the modification of the natural bacterial receptorstructure it is preferred to perform the substitution thereof byresorting to a genetic engineering, such as site-directed mutagenesis.

With regard to the interaction partner of the modified natural bacterialreceptor a multitude of substances are conceivable, such as proteins,lipids, carbohydrates and inorganic substances. Among carbohydratesexamples are blood group determinants and pathogen specificoligosaccharides.

In regard to proteins conceivable interaction partners are IGF-I,IGF-II, hGH, Factor VIII, insulin and apolipoprotein and theirrespective receptors as interaction partners. Furthermore, by selectingnew receptor variants with specificity for different folding forms ofproteins, affinity resins are analytical tools to facilitate theisolation of correctly folded molecules can be produced. Furtherexamples are viral coat proteins, bacterial antigens, biotin and cellmarkers, such as CD 34 and CD 4.

Although the present invention is applicable to a variety of naturalbacterial receptors the following illustration of the invention more indetail will be directed to the use of the IgG-binding domains Z, C1 andB2A3. The concept of the present invention residing in the use ofartificial bacterial receptors based on the natural structures ofnaturally occurring bacterial receptors is associated with severaladvantages. Thus, the invention makes it possible to use robust andstable, highly soluble and secretion competent receptors. This is incontrast to previous techniques based on the use of polyclonals andmonoclonals, such as for diagnostic purposes, which are not very stablein connection with storage, varying conditions, such as varyingtemperatures etc. Furthermore, the invention makes it possible to modifynatural bacterial receptors to obtain desired interaction capacities forspecific purposes.

For the selection of such functional variants in a large repertoire, apowerful selection system must be employed. Recent developments in thisfield offer alternative methods. One of the most important tools forprotein engineering that has emerged during the last years is the phagedisplay of proteins. By recombinant DNA techniques, single phageparticles can be prepared which on their surface carries a protein fusedto a phage-coat protein. By panning from a large pool of phages bearingdifferent proteins, or variants of a specific protein, specific phageclones can be sorted out, which displays a certain bindingcharacteristic WO92/20791 to Winter et al!. Since the phage particlecontains packed DNA encoding the phage protein components, a couplingbetween the specific variant of the displayed protein and thecorresponding genetic information is obtained. Using this technique,typically 10⁹ phge clones can simultaneously be generated and subjectedto panning for screening of desired characteristics. The phage displaytechnique can be used for selection of both small peptides as well asmore complicated proteins such as antibodies, receptors and hormones.For selection of proteins which cannot be secreted, which is aprerequisite for phage display, intracellular systems have beendeveloped in which the library of proteins are fused to a repressorprotein with affinity for a specific plasmid-borne operator regionresulting in a coupling between the specific protein variant and theplasmid that encoded it. An alternative to the phages as bearer ofprotein libraries would be to use bacterial cells. Recently, display ofrecombinant proteins on the surface of Staphylococcus xylosus based onfusions to the cell-wall anchoring domain was demonstrated, which opensthe possibility of display also of repertoires of proteins for affinityselection of specific variants Hansson, M. et al 1992 J. Bacteriology174:4239-4245!. Furthermore, by structure modelling using computergraphic simulations, predictions of the binding and function of alteredvariants of a protein can theoretically be done before the constructionof the gene encoding the protein.

As indicated above the present invention describes the construction ofnovel proteins based on the mutagenesis of surface exposed amino acidsof domains derived from bacterial receptors. These artificial bacterialreceptors can be selected for different applications using a phagedisplay system. The benefits from using bacterial receptors asstructural frameworks are several. They have evolved to express abinding function without disturbing the overall structure. They arenaturally highly soluble, robust to unphysiological conditions, such aspH and heat, folding efficient and are in addition secretion competent.

The invention finds use in several different areas.

The introductory part of the above-identified patent specificationWO92/20791 gives an excellent survey on antibodies and their structure.Reference is particularly made to page 1 thereof.

The bacterial receptors SPA and SPG have been widely used in antibodytechnology for detection and purification of antibodies from e.g.hybridom supernatants and ascites fluids. However, not all antibodiesare recognized by these receptors, depending on species and subclass.For the smaller subfragments of antibodies (FIG. 4), SPA and SPG show alimited binding, and efficient tools for general purification schemesare lacking. However, from a repertoire of mutant receptors includingSPA and SPG, forms displaying a broader affinity for antibodies andsubfragments thereof can potentially be selected.

The complex structural organization of antibodies has a number ofconsequences for their use in different applications as well for theproduction of recombinant derivatives. For use in immunosorbents, thearrangement of subunits connected by disulphide bonds can lead to aleakage of released heavy and light chains from columns. The requirementof successful docking of the two subunits contributing to the antigenbinding site makes the production in bacterial of small subfragmentswith a low association difficult. The folding of the antibody isdependent on the formation of intra- and inter chain disulphidebonds,which are not able to form in the intracellular environment of bacterialcells. High-level intracellular expression systems for recombinantantibodies leads to the formation of inclusion bodies, which have to berenatured to obtain biological activity. These limitations make itworthwhile to search for alternatives for use as protein domains capableof specific binding, to replace antibodies in a vast number ofapplications.

The CDR regions forming the antigen binding part of an antibody forms atotal surface available for the antigen of approximately 800 Å², withtypical 10-20 residues from the antibody involved in the binding. Usingthe structure of the complex determined by X-ray crystallography betweenan individual domain B of SPA and human fc IgGI! as a starting pointabout 15 amino acids of the said domain involved in this binding can bedetermined or postulated. The binding surface of about 600 Å² is of thesame order of magnitude as between an antibody and its antigen. Byarbitrary in vitro mutagenesis of these positions simultaneously thereis obtained a large library of Z variants with modified functionalproperties. In view of the fact that the regions of the Z domainconstituting the very stabile so called three-helix bundle is maintainedin its native form spectra of proteins are generated which could beconsidered as "artificial antibodies" and which have the expected highsolubility and excellent folding properties capable of binding to alarge number of new ligands. Fusions of these artificial receptors toconstant regions can be constructed to recruite effector functions, suchas complement binding or triggering of ADCC (antibody dependent cellularcytotoxicity).

There are several potential advantages of utilizing the SPA structure Z!as a starting point for such "artificial antibodies" or artificialbacterial receptors. For a period of about 10 years a large number ofproteins have been produced as fusions to SPA, where on has utilized theunique properties of the fusion partner in expression, refolding andpurification. In these applications the Z domain has been found to beextremely soluble, stable against proteases, easy to produce in largeamounts and foldable to a correct structure also intracellularly inEscherichia coli (no cysteins). Immunoglobulins (Ig:s) are substantiallytetramers built up from so called β-sheet structures which stabilize theorientation of the antigen-binding loops which in turn consist ofcontinuous peptide sequences. This is to be compared to the monomeric Zdomain built up from so called three-helix bundle consisting of threeclosely packed α-helix structures, where the Fc-binding amino acids arefound discontinuously in the sequence but in the folded protein arepositioned on one and the same binding surface. This difference withregard to the structural elements contributing to the formation of thebinding surface enables new possible conformations which cannot beobtained in natural antibodies. The ability of Z to be folded to thenative structure also under conditions prevailing in the site ofcytoplasma opens the possibility of using also derivatives thereofclinically. Genes coding for artificial antibodies with for examplevirus-neutralizing capacity can be distributed to cells through socalled gene therapy resulting in interrupting the infection at an earlystage.

From structure data for the complex between an individual Ig-bindingdomain C1! of SPG and human Fc the binding surface can be studies. Thebinding which is essentially of an electrostatic nature involves sidechains from amino acids from the α-helix as well as from the subsequentβ-sheet #3!. These differences in structure compared to the Z domainmakes it useful also to create a library of C1 variants to investigatewhether differences in binding patterns for artificial antibodies can beobserved depending on the different conditions as regards the topologyof the binding surface. Repertoires based on the structures of these andother receptors therefore offer different possibilities in the creationof artificial forms with novel functions.

When producing recombinant proteins the purification of the product isfrequently a major problem. By expressing the target protein as a fusionto a so called affinity tail the hybrid product can effectively andselectively be recovered from the cell lysate or in certain cases fromthe culture medium by passage through a column containing an immobilizedligand. Several such gene fusion systems have been described which arebased on the interaction of a certain protein with a ligand. Forindustrial applications it is often desirable to clean effectively thecolumns between the runs to satisfy purity requirements by authorities.Depending on the nature of proteins the relatively harsh conditions(NaOH, acids, heat) often used for organic or physical matrises, forexample in ion exchange chromatography and gel filtration, can normallynot be used. Here the use of new ligands based on stable structuresoriginating from bacterial receptors are of great importance. In thisconnection the Z domain from SPA is an excellent example since saiddomain can be subjected to such difficult conditions as a pH of 1 orheating to 80° C without denaturating non-reversibly (see Example 2below). From the library of for example Z variants interesting proteinproducts can be selected for use immobilized on a solid phase foraffinity chromatography. These protein ligands are resistant toeffective purification conditions and are therefore useful repetitivelyon a large scale. In traditional immuno affinity chromatography whereimmobilized monoclonal antibodies are used for the selectivepurification of a certain product there are problems with leakage fromthe column of subunits (heavy and light chains) of the antibody since itconsists of four polypeptide chains linked by cystein bridges. Since theartificial bacterial receptors consist only of one polypeptide chainthis problem will be avoided. One particular area of interest isselection for binding to carbohydrates. Lectins, nature's binders tothis large and important group of biomolecules, have been found to bedifficult to purify and have limited stability. Since the generation ofantibodies against carbohydrates has been found to be quite complicateda selection of new artificial lectins will be of great importance toresearch, diagnostics and therapy.

In the production of recombinant proteins in bacterial hostsprecipitates of the gene product are frequently formed, so calledinclusion bodies. In order to obtain a native structure of the proteinthis must be subjected to refolding in vitro. A limitation in suchprocess one is often confronted with is the fact that a great part ofthe material precipitates in the procedure which results in low yields.By producing the protein with an extension in the form of either a shorthydrofilic peptide or an easily soluble complete domain Samuelsson, E.et al 1991 Bio/Technol. 9:363-366! substantially higher concentrationsof the protein will be obtained without precipitation taking placeduring renaturation. For example the high solubility of the said domainenables the use of increased solubility of proteins in either refoldingfrom inclusion bodies or in so called reshuffling of disulphide bridges.From libraries of artificial receptors new forms can be selected havingimproved properties to facilitate and even make refolding of recombinantproteins possible (cis-acting chaperones).

Recently a new unit operation for the purification of recombinantproteins based on ion exchange chromatography in so called expanded bedhas been described Hansson, M. et al 1994 Bio/Technol. in press!. Inthis connection there is used a difference in isoelectric point betweenthe target protein and the proteins of the host cell for selectiveenrichment on a positively charged ion exchange matrix. By fusion to theacid Z domain (pI 4.7) the ion exchange step can take place at a pH, atwhich the majority of the contaminants were of the opposite chargecompared to the fusion protein. By constructing libraries of bacterialreceptors where a selection of amino acids have been replaced by theacid amino acids aspartate and glutamate also very acid and solubilityincreasing domains can be produced for use as fusion partners in theproduction of recombinant proteins.

As previously described affinity systems based on protein ligands arenot totally suitable for industrial purposes in view of the harshconditions required in the cleaning of columns. Therefore, there is aneed for fusion partners having specific affinity towards simple andcheap organic ligands. Panning of phage display libraries of differentbacterial receptors against such ligands provide novel affinity tailssuitable for the use as fusion partners for the production purificationof recombinant proteins.

The present invention provides means for producing and selectingproteins with novel functions. According to the invention this isachieved by extensive mutation of defined residues of stable domains ofbacterial receptors. Due to the novel functions of the artificialbacterial receptors, these can be used as specific binders fortherapeutic, diagnostic, biotechnology or in research.

The present invention will now be described more in detail by specificexamples with reference to the appended drawings. In the drawings:

FIG. 1 A. Schematic representation of staphylococcal protein A showingthe signal peptide (S), five IgG-binding regions E-D-A-B-C!, followed bycell wall anchoring region X-M!. B. Computer graphic representation ofthe complex between domain B from SPA and human Fc1 determined by X-raycrystallography. Note that the third helix of SPA is not seen in thisfigure.

FIG. 2 A. Schematic representation of streptococcal protein G from thestrain G148 showing the signal peptide (Ss), region E (E), therepetitive serum albumin binding A-B region, the spacer region (S),followed by the IgG binding domains C1 through C3, spaced by the Dregions and finally the cell wall anchoring region W. B. Computergraphic representation of the complex between domain C1 of SPG and humanFc1 determined by X-ray crystallography.

FIG. 3 Schematic representation of the three helix bundle structure ofthe 58 residue SPA analogue Z. Indicated are some of the side chainsproposed to be involved in the binding to Fc with the exception of F30,which stabilizes the helix-helix packing.

FIG. 4 IgG antibody structure, showing the different subfragmentsFab,Fd,Fc and the scFv composed of the VH and VL connected by a short(ca 15 aa) linker.

FIG. 5 A. General concept for the gene assembly strategy used for thegeneration of the Z gene libraries. For the construction of the libraryof acid Z derivatives, only resides 9, 11, 14, 27 and 35 were alteredusing the degenerated oligonucleotides ACID-1, ACID-2. The PCR primersused for the amplification of the assembled library were ZLIB-3 (PCRprimer 5') and ZLIB-5 (PCR primer 3'). B. The PCR products from theamplification of the assembled library encoding 46 of the 58 residues ofthe Z-domain can be cloned into phagemid DNA harboring the remainingC-terminal part of Z. This gene is fused in frame with the gene forprotein III of the M13 family of E. coli bacteriophages. This enablesthe display on the phage surface of the repertoire of acid Z-variants.

FIG. 6 Oligonucleotides used for the construction of Z-libraries. Forthe library of acid Z-variants described in Example 2, onlyoligonucleotides ZLIB-1, 2, 3, 4, 5, LONGBRIDGE, ACID-1 and ACID-2 wereused.

FIG. 7 DNA sequences of clones derived from the acid Z protein library.Bold figures indicate amino acid positions in the Z-domain. For claritythe positions of the restriction sites Acc I and Nhe I are shown.

FIG. 8 Result from analysis of the temperature stability of anindividual Z domain at pH 2.9. The content of α-helicity in the samplewas monitored by measuring the ellipticity at s222 nm during atemperature scan.

FIG. 9 Phagemid vector pKN1. The library PCR products encoding thevariegated helices 1 and 2 (both the acid and the extensive library) wassubcloned into the phagemid vector, pKN1, containing the gene forresidues 44-58 of the wild type Z domain (essentially helix 3), followedby the gene for a 46 residues serum albumin binding region (ABP) derivedfrom streptococcal protein G linked in frame with a truncated version ofthe M13 phage coat protein 3 gene. The phagemid contains the origin ofreplication derived from plasmid pBR322 as well as the intergenic region(fl ori) required for packing into the phage particles.

FIG. 10 SDS-PAGE. HSA-affinity purified proteins from the periplasm ofEscherichia coli cells producing the wild type Z domain and twodifferent acid Z-variants as ABP fusion proteins encoded from theirrespective phagemid vectors were analyzed by SDS/PAGE. M, molecularweight marker; lane 1, wild type Z domain; lane 2, clone 10; lane 3,clone 12.

FIG. 11 CD-data. Overlay plot of CD spectra obtained for the wild type Zdomain and two variants of the Z-protein library. The signals of theproteins were obtained after subtraction of the CD signal contributionof the ABP tail, present during the analysis.

FIG. 12 Ion exchange chromatography. The two acid Z-variant proteins no.10 and no. 12 together with the wild type Z-domain (produced as ABPfusion proteins) were each subjected to analysis at pH 5.5, employing ananion exchange chromatography column. Elution of the proteins from thecolumn was obtained by a NaCl gradient. Top: acid Z-variant no. 12;middle, acid Z-variant no. 10; bottom, Z (wild type). Note that the wildtype Z protein was not retarded on the column at this pH.

FIG. 13 Z-domain structure. A main-chain trace representation of themodel of the structure of the native Z-domain. The structure of helicesone and two are from the co-crystal structure between domain B of SPAand Fc (Deisenhofer, (1981) Biochemistry, 20, 2361-2370). The thirdhelix was built based on the secondary structure assignments from NMRspectroscopy (Gouda et al., (1992) Biochemistry, 31, 9665-9672).Non-hydrogen atoms of side-chains of residues that were mutated in theconstruction of the combinatorial library are displayed asball-and-stick models. The display was generated by the programMOLSCRIPT (Kraulis (1991) J. Appl. Cryst., 24, 946-950).

FIG. 14 Amino acid sequences. Result from DNA-sequencing of 31 randomlychosen Z-variants from the library. The residues subjected to themutagenesis are boxed. Horizontal lines indicate nucleotide identitywith the wild type Z sequence listed at the top. Indicated are theclones that were expressed and characterized as fusion proteins to theABP-tail.

FIG. 15 Aminoacid distribution. Result from the statistical analysis ofthe deduced amino acids at the mutated positions. In total, 13 residuesfrom 31 clones (403 codons) were included in the calculation. The ratiosbetween observed and expected frequencies are shown for all 20 aminoacids as well for the only termination signal (TAG) included in theNNG/T degeneracy profile.

FIG. 16 SDS-PAGE analysis. HSA-affinity purified proteins from theperiplasm of E. coli cells producing the wild type Z domain and fourdifferent Z-variants as ABP fusion proteins encoded from theirrespective phagemid vectors were analyzed by SDS/PAGE. Lanes 1-5:Reduced conditions. Lanes 6 and 7: Non-reduced conditions. Lane 1, wildtype Z domain; lane 2, clone 16; lane 3, clone 21; lane 4, clone 22;lane 5, clone 24; M, molecular weight marker; lane 6, clone 16 and lane7, clone 22.

FIG. 17 CD-data. Overlay plot of CD spectra obtained for the wild type Zdomain and four variants of the α-helical protein surface library. Thesignals of the variants were obtained after subtraction of the CD signalcontribution of the ABP tail, present during the analysis.

FIG. 18 Biosensor assay. An overlay plot of sensorgrams obtained fromthe BIA-core™ analysis of the wild type Z domain and four differentvariants (no. 16,21,33,24; FIG. 4) fused to the ABP tail. TheIgG-binding activities of the different proteins were analyzed using asensor chip coated with approx. 5000 RU human polyclonal IgG andinjections of 45 μl pulses at 2 μl/min of 1500 nM solutions of thedifferent proteins. Note that the differences in plateau values ofsignals during the injections of the variants no. 16,21,22 and 24 is dueto divergent dilutions into the driving buffer.

All reagents and DNA constructions are available at the department forBiochemistry and Biotechnology, Royal Institute of Technology,Stockholm, Sweden.

Material

The oligonucleotides (FIG 6) were purchased from Scandinavian GeneSynthesis (Sweden), and phosphorylated where indicated according toManiatis et al (1988) Molecular cloning. A laboratory manual. ColdSpring Harbor Laboratory Press!. ZLIB-1 was biotinylated in the 5'-endenabling immobilization on paramagnetic beads M-280 Streptavidinpurchased from Dynal A/S (Norway). Washing/binding buffer was 1 M NaCl,10 mM Tris-HCl, pH 7.5, 1 nM EDTA (ethylenediamine tetraacetic acid).The annealing/ligation buffer was 30 mM Tris-HCl, pH 7.5, 10 mM MgCl₂,0.2 mM ATP, 1 mM 1.4 dithiothreitol (DTT). DNA ligase were fromBoehringer Mannheim, Germany. 10×PCR buffer contained 20 mM MgCl₂, 2 mMdNTPs, 100 mM Tris-HCl, pH 8.3, 50 mM KCl, 1% Tween 20. Taq DNApolymerase was from Cetus Inc., USA. The thermal cycler was aPerkin-Elmer 9600. For the temperature/stability scanning a J-720spectropolarimeter (JASCO, Japan) was used. Escherichia coli strainRR1ΔM15 Ruther, U. (1982) Nucl. Acids Res. 10:5765-5772! prepared forcompetence Maniatis et al (1988) Molecular cloning. A laboratory manual.Cold Spring Harbor Laboratory Press! was used as host for thetransformation. Agar plates contained 100 μg/ml of ampicillin.

EXAMPLE 1

Construction of an acid Z-library

The synthetic 58 residue SPA analogue Z Nilsson et al, Prot. Eng! wassubjected to a mutagenesis approach to construct new variants with analtered pI, in order to produce fusion partners for recombinant proteinsto be purified by ion-exchange chromatography. Based on the crystalstructure of the complex between the B-domain of SPA and human FclDeisenhofer, J. et al 1981, Biochemistry 20:2361-2370!, five residuesfrom the B-domain participating in the binding were chosen as targetsfor mutagenesis. These five codons corresponding to the Z-residues No.9, 11, 14, 27 and 35 positioned in helices 1 and 2 were alteredsimultaneously using degenerate oligonucleotides with the tripletsequence G(C/A)(C/A) at these positions resulting in the codons for theamino acids alanine (50%), aspartic acid (25%) and glutamic acid (25%),respectively. Using a solid phase gene assembly strategy Stahl et al,Biotechniques 14:424-434! a library of genes encoding 3⁵ (243) acidicvariants of the synthetic IgG-binding Z-domain was created (FIG. 5).Twenty microlitres (200 μg) of paramagnetic streptavidin-coated beadswere washed with washing/binding buffer and incubated with 15 pmole ofpre-hybridized oligonucleotides ZLIB-b (biotinylated) and ZLIB-2, for 15min at RT at a final volume of washing/-binding buffer of 40 μl. Afterligation and washing, approximately 15 pmole each of theoligonucleotides ACID-1 (degenerated), LONGBRIDGE, and ACID-2(degenerated) and the preannealed linker pair ZLID-4/ZLIB-5 were addedin a repetitive manner, with intervening washing steps according toStahl et al Biotechniques 14:424-434!. After completed assembly, thedifferent fragments were ligated for 15 min at 37° C. To amplify theamount of DNA coding for the Z(Acid)-library still immobilized onto thebeads, a fraction was withdrawn and subjected to PCR. The PCR mix (50μl) contained one pmole each of PCR primers ZLIB-3 and ZLIB-5, 5 μl eachof the ligation mix, 10×PCR buffer and 10×CHASE, 1 unit of Taqpolymerase and sterile water to 50 μl. The temperature cycling programmewas: 96° C., 1 min, 60° C., 1 min and 72° C., 2 min, repeated for 35cycles. Analysis by 1% agarose gel electrophoresis showed a band of theexpected size of 179 bp band from the PCR of the Z(Acid)-library, wascut out from the gel and purified (Geneclean™, Bio 101, Inc. USA) priorto insertion in a plasmid vector (TA-cloning™ kit, Invitrogen, Inc. USA)suitable for solid phase DNA sequencing Hultman et al, 1988!. Aftertransformation and spreading on ampicillin containing agarplates twocolonies were chosen for analysis of the obtained sequences. The results(FIG. 6) show that the expected degeneracy was found at the desiredpositions.

EXAMPLE 2

Measurement of the temperature stability of the Z conformation

The temperature stability of the Z conformation was determined byfollowing the ellipticity at 222 nm by circular dichroism (CD)spectroscopy through a temperature scan. The wavelength is used tomonitor the presence of α-helicity of Z Cedergren et al. 1993 Prot. Eng.6:441-448!. The experiment was performed at rather low pH (approximately2.9) in order to destabilize the molecule since the mid-point oftemperature denaturation (Tm) is ˜95° C. at neutral pH (data not shown),which is outside the range that can be determined by a complete scanthrough the transition under normal atmospheric pressure. The experimentshows (FIG. 4) that the Tm (as defined by the inflexion point of thetemperature scan) of the Z domain is as high as 71° C. at pH 2.9. Thisdemonstrates the extreme temperature stability of the α-helices of the Zmolecule.

The experiment was performed in a J-720 spectropolarimeter (JASCO,Japan) and the temperature was controlled by circulating water throughthe cuvette holder from a NESLAB water bath. The temperature wasmonitored in the cuvette through a micro sensor device (JASCO, Japan).The buffer was 50 mM acetic acid, pH 2.9. The protein was domain ZCedergren et al 1993 Prot. Eng. 6:441-448! at a protein concentration of50 μg/mL and the cuvette cell path length was 1 cm. The temperature scanspeed in the experiment was 50° C./h.

EXAMPLE 3

Characterization of proteins derived from the acid Z-library

Two protein variants derived from the acid Z-library were expressed inEscherichia coli, purified and characterized using SDS-PAGE, circulardischicism fusion exchange chromatography. The PCR products from a solidphase gene assembly (see example 1) were restricted with 45 U Esp 3I(Labassco AB, Sweden) and 50 U Nhe I (Pharmacia, Sweden) in 200 μlbuffer (33 mM Tris-acetate, pH 7.9, 10 mM Mg-acetate, 66 mMpotassium-acetate, 0.5 mM DTT and 0.1 mg/ml BSA). The mix was overlaidwith mineral oil and incubated at 37° C. over night. The restrictedfragments (approximately 5 μg) were purified byphenol/chloroform/isoamylalcohol extraction followed by additionalwashing with chloroform and later ethanol precipitated before ligationat 15° C. over night to Mlu I-Nhe I cleaved pKN1 vector (1 μg) (seebelow) using 13.5 Weiss units of T4 DNA ligase. The ligation mixture washeat-treated at 70° C. for 20 min, extracted withphenol/chloroform/isoamylalcohol followed by washing with chloroform,ethanol precipitated and redissolved in 20 μl of sterile water.

The phagemid vector pKN1 (FIG. 9) was constructed in several steps asfollows. A double stranded linker encoding the invariant residues 44-58of the Z-domain was formed from oligonucleotides ZLIB-6 and ZLIB-7 andcloned as a Mlu I-Xho I fragment into phagemid pKP986 (A kind gift fromDr. Lars Abrahmsen, Pharmacia BioScience Center, Sweden), resulting inpKN. Plasmid pKP986 encodes the E. coli Omp A leader peptide followed byresidues 249-406 of fd filiamentous phage coat protein 3 (Lowman et al.(1991) Biochemistry, 30, 10832-10855) under the control of a lacpromoter. A gene fragment encoding a monovalent serum albumin bindingregion derived from streptococcal protein G was amplified by PCR fromthe plasmid pB2T (Eliasson et al., Molecular Immunol., 28, 1055-1061),using primers ABP-1 and ABP-2 (which contain Xho I and Sal I recognitionsites, respectively) and cloned into Xho I restricted plasmid pKN,yielding pKN1. This phagemid vector thus encodes for the Omp A signalpeptide, the third helix of the wild type Z domain followed by a 46residue albumin binding protein (ABP) linked to residues 249-406 of fdphage protein III and is adapted for insertion of Esp 3I/Nhe I-digestedPCR products encoding variegated helices one and two of the Z domain.

Freeze competent E. coli RR1ΔM15 (supE44 lacY1 lacZ ara-14 galK2 xyl-a5mil-1 leu56 proa2ΔimrcC-mrr) recA⁺ rpsL20 thi-1 lambda⁻ FlaclqlccZΔM15!) (Ruther, 1982) Nucleic Acids Research, 10, 5764-5772)cells were transformed with the ligation mixture according to Maniatisand coworkers (Maniatis et al. (1982) Molecular cloning: A LaboratoryManual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press) andplated on agar plates containing 100 μg/ml ampicillin Sigma, USA) and 1%glucose. Small amount of cells from randomly picked colonies wereseparately subjected to two-step PCR amplifications (30 cycles: 96° C.,15 s: 72° C., 2 min) on a GeneAmp PCR System 9600 (Perkin Elmer, USA),using 5 pmoles of primers RIT-27 and NOKA-2 (biotinylated) in 20 mM TAPS(pH 9.3), 2 mM MgCl₂, 50 mM KCl, 0.1% Tween 20, 0.2 mMdeoxyribonucleoside triphosphates (dNTPs) and 1.0 U of Taq DNApolymerase (Perkin-Elmer). The solid-phase DNA sequencing of the PCRproducts was performed employing the FITC labeled sequencing primersNOKA-3 (for the immobilized strand) and ABP-2 (for the eluted strand) ona robotic workstation (Biomek™ 1000, Beckmand Instruments, Fullerton,Calif.) and an Automated Laser Fluorescent (A.L.F.) DNA Sequencer™(Pharmacia Biotech, Sweden) as described by Hultman and coworkers(Hultman et al., (1989) Nucleic acids Research, 17, 4937-4946).

Two clones with the different encoded acid aminoacid substitutions (boldface) at the positions 9, 11, 14, 27 and 35 in the Z-domain according totable 1 were selected for further analysis. The wild type Z domain andthe two different acid Z-variant proteins (clones no. 10 and 12) wereexpressed from their respective phagemid vectors as fusions to the serumalbumin binding tail (ABP) and purified by human serum albumin-affinitychromatography

                  TABLE I                                                         ______________________________________                                        Amino acid substitutions for selected clones in the acid                      Z-library.sup.a.                                                                     Encoded amino acid at position no.                                     Clone no.                                                                              9         11     14      27   35                                     ______________________________________                                        w.t.     Gln       Asn    Tyr     Arg  Lys                                    10       Glu       Asp    Asp     Ala  Glu                                    12       Glu       Asp    Asp     Ala  Ala                                    ______________________________________                                         .sup.a.Letters in bold face indicate acid aminoacids                     

Colonies of E. coli RR1ΔM15 cells harbouring the corresponding phagemidvectors were used to inoculate 100 ml of Tryptic Soy Broth (Difco),supplemented with ampicillin (100 μg/ml). The cultures were grown at 37°C. to an OD_(600nm) =1, followed by induction with a final concentrationof 1 mM IPTG and incubation at 30° C. over night. The cells wereharvested by centrifugation at approximately 5000 g for 10 min andperiplasmic proteins released by osmotic shock. The periplasmic contentfrom the cells were subjected to affinity chromatography onHSA-Sepharose as described by Nygren and coworkers (Nygren et al.,(1988) J. Mol. Recognit., 1, 69-74) and analyzed by SDS/PAGE on amonogeneous 12% slab gel (BioRad Inc., USA), which was stained withCoomassie Brilliant Blue R-250. For all proteins appr. 1.5-2.5 mg/Lculture could be recovered, indicating similar production and secretionefficiencies for the variants and the wild type domain. In addition, theresults from the SDS-PAGE analysis (FIG. 10) of purified proteinssuggest that the acid Z variants analyzed are stably expressed in E.coli.

To investigate if the secondary structure content of the derivatives waspreserved after the surface mutagenesis, a subtractive circulardichroism analysis was performed. IgG- or HSA- affinity chromatographypurified proteins Z. Z-ABP, the acid derivatives no. 10 and 12 fused tothe ABP tail as well as the ABP-tail itself were subjected to a 250 to184 nm (far UV) circular dichroism analysis at room temperature using aJ-720 spectropolarimeter instrument (JASCO, Japan). The scanning speedwas 10 nm/min. The cell pathlength was 1 mm. Solutions (approximately0.1 mg/ml) of the different proteins were prepared in 20 mM phosphatebuffer pH 6.5, supplemented with 0.05% Tween 20 (Kebo AB, Sweden).Accurate protein concentrations were determined by amino acid analysison a Beckmann 6300 amino acid analyzer equipped with System Gold datahandling system. CD signals for the derivatives were obtained bysubtracting the signal obtained for the ABP tail, after adjustments fordifferences in protein concentrations, followed by normalization foramino acid contents.

A comparison of signals obtained from 250 to 184 nm for the wild Zdomain and the acid variants fused to the ABP-tail was performed aftersubtraction of the contribution from the ABP tail itself. The resultshows that for the two acid Z-derivatives, spectra similar to the wildtype Z domain were obtained with a characteristic minimum at 208 nm andan inflexion point at 222 nm (Johnson, 1990) (FIG. 11). This suggeststhat the three helix bundle framework is preserved in these mutants.

The two Z-variants, no. 10 and 12, contain four and three introducedacid aminoacids, respectively, compared to the native Z domain. In orderto investigate if the introduced acidity was reflected as differences intheir isoelectric points, they were subjected to a gradient elution froman anion exchange column. The proteins Z(wild type) and the acidvariants no. 10 and no. 12 (all produced as ABP fusion proteins) wereeach (5 μg) dissolved in 300 μl of 20 mM Piperazine buffer (pH 5.5) andseparately applied at 100 μl/min on a MonoQ, PC 1.6/5 column (Pharmacia,Sweden). Elution of the proteins were performed by applying a NaClgradient in Piperazine buffer (pH 5.5) (Sigma, USA) ranging from 0-50%NaCl in 20 min. The results from the analysis (FIG. 12) shows that thetwo acid Z-variant proteins were eluted at different NaCl concentrationssuggesting clear differences in isoelectric points. In contrast, at thepH chosen during the experiments, the wild type Z-domain did notinteract with the resin, and was therefore seen in the flow-through.

Thus, the series of experiments performed on the two acid Z-variantproteins shows that the expression behaviour, proteolytic stability andsecondary structure content of the variants were unchanged when comparedto the native Z-domain. Furthermore, a novel functions were introducedinto the two Z-variants by the substitution of surface located positionswith acid amino acids. The two acid variants can be used e.g. as fusionpartners to facilitate purification of recombinant proteins by ionexchange chromatography at low pH. Thus, it is showed that among themembers of the acid Z-library, variants with novel functions can beisolated.

EXAMPLE 4

Construction and characterization of a combinatorial library ofZ-variants

A library of Z-variants was assembled using a solid-phase gene assemblystrategy (see example 1). Most of the amino acid residues suggested totake part in the binding to Fc (Deisenhofer, (1981) Biochemistry, 20,2361-2370) were found to be on the molecule surface (Q9, Q10, N11, F13,Y14, L17, N28, Q32 and K35), and therefore included in the mutagenesis.In addition, based on their surfacial location, other residues (H18,E24, E25 and R27) were also decided to be included. In total, 13residues in the Z scaffold where thus chosen for simultaneous and randommutagenesis. A set of oligonucleotides (FIG. 6) were synthesized forconstruction of the library of surface mutants of the 58-residuesmonovalent IgG-binding domain denoted Z. In this library, the codons forQ9, Q10, N11, F13, Y14, L17 and H18 located in the first α-helix andE24, E25, R27, N28, Q32 and K35 in the second α-helix of the Z domain(FIG. 13) were substituted for degenerate NNK (K=G cr T) codons using asolid phase strategy utilizing the single stranded degenerateoligonucleotides for the assembly. The chosen NNK degeneracy includes 32codons covering all 20 amino acids, including the TAG (amber)termination signal.

Oligonucleotide ZLIB-1 was synthesized with a 5' biotin group to enablerobust anchoring onto streptavidin-coated paramagnetic beads used assolid support during the gene assembly. This ZLIB-1 oligonucleotide,together with its complementary sequence (ZLIB-2) encodes residues 1-8of the Z domain, preceeded by the first six residues of region E ofprotein A which were included to facilitate the E. coli secretion of theZ variants (Abrahmsen et al., (1986) EMBO J., 4, 3901-3906). Theoligonucleotides DEGEN-1 and DEGEN-2 (Table I) encode the two mutatedhelices of the Z domain, respectively, normally involved in Fc-binding.Theoretically, full and simultaneous NNK degeneracy at the 13 selectedpositions would yield a combinatorial library of appr. 8-10¹⁶ proteinvariants encoded by 3.7-10¹⁹ different DNA sequences. However, here theassembly of the library was initiated by the immobilization of appr. 15pmole of prehybridized oligonucleotides ZLIB-1 AND ZLIB-2 (FIG. 6),which limits the theoretical size of the Z-library to appr. 0.9-10¹³different DNA sequences encoding appr. 2-10¹⁰ Z variants. The assemblywas continued by the addition and ligation of a preformed constructobtained after ligation of equimolar amounts of oligonucleotides DEGEN-1and DEGEN-2, facilitated by the bridging oligonucleotide BRIDGE (FIG.6).

To complete the assembly, a fragment consisting of the prehybridizedoligonucleotides ZLIB-4 and ZLIB-5 was added to the beads for ligation.This fragment encodes the second loop and the first six residues of theunaltered third helix of the Z domain. After completed assembly,oligonucleotides ZLIB-3 and ZLIB-5, containing the recognition sequencesfor the endonucleases Esp 3 I and Nhe I respectively, were used asprimers for PCR amplification of the assembled constructs using onetenth of the bead-immobilized ssDNA as template (theoreticallycorresponding to 2-10⁹ protein variants). To avoid unwanted interferenceduring the amplification, oligonucleotides ZLIB-2, BRIDGE and ZLIB-5were first eluted with alkali. The resulting PCR product was analysed byagarose gel electrophoresis and found to be homogenous and of theexpected size, 179 bp.

The PCR product was subcloned into the pKN1 phagemid vector containingthe gene for residues 44-58 of the wild type Z domain in frame with atruncated version of the fd phage coat protein 3 gene for surfacedisplay on phage particles upon helper phage superinfection of phagemidtransformed E. coli cells (Lowman et al., (1991) Biochemistry, 3010832-10844) (FIG. 9). In addition, the phagemid vector contains aninterspaced in-frame cassette encoding a 5 kDa (46 aa) serum albuminbinding region (denoted ABP) derived from streptococcal protein G(Nygren et al., (1988) J. Mol. Recognit., 1, 69-74; Nilsson et al.,(1994) Eur. J. Biochem., 224, 103-108), enabling efficient affinitypurification of produced Z variants devoid of their native Fc-bindingactivity. Furthermore, the serum albumin binding activity canpotentially be used for pre-selection of phage particles carryingrecombinant molecules, prior to the panning for Z variants with newbinding functions, to decrease the background originating fromunspecifically bound non-recombinant phage particles.

After transformation, PCR screening (using the oligonucleotides RIT-27and NOKA-2) of 25 clones showed that over 95% (24/25) of the clonescontained an insert of the expected length, suggesting that the geneassembly procedure was carried out with high efficiency. Fortyfivetransformants were randomly selected and subjected to direct solid phaseDNA sequencing (see Example 3) in order to further analyze the qualityand heterogeneity of the library. Approximately 69% of the clones werecorrect, containing wild type and degenerate codons at expectedpositions. The remaining clones had spurious discrepancies which in partcan be attributed to the oligonucleotide synthesis or errors introducedduring PCR. The correct clones (31 clones) (FIG. 14) were furtheranalyzed for codon representation at the 13 degenerate positions. Thedistribution of the total 403 resulting deduced amino acids among the 32codons included in the NNK degeneracy profile shows a close correlationwith the expected frequencies for these yet unselected clones (FIG. 15).To investigate the expression and stability of the Z-variants, fourclones (no. 16, 21, 22, 24: FIG. 14) with different degrees ofsubstitution as well as the wild type Z domain were produced as ABPfusions encoded from their respective phagemid vectors. Soluble proteinsfrom the periplasm of IPTG-induced cultures were subjected toHSA-affinity chromatography employing the ABP-tail for general andefficient recovery (Nygren et al., (1988) J. Mol. Recognit., 1, 69-74).For all proteins appr. 1.5-2.5 mg/L culture could be recovered,indicating similar production and secretion efficiencies for thevariants and the wild type domain. The results from a SDS-PAGE analysis(FIG. 16) of purified proteins suggest that the four Z variants analyzedare stably expressed in E. coli. The smaller band with HSA-bindingactivity, seen with different intensities most probably correspond tothe ABP-tail itself (5 kDa), resulting from proteolytic cleavage betweenthe Z variant and the ABP tail. Interestingly, both Z-variants (no. 16and 22) with introduced cysteine residues formed dimers, which could beobserved under non-reducing conditions during SDS-PAGE (FIG. 13; lanes 6and 7).

To investigate if the secondary structure content of the derivatives waspreserved after the extensive surface mutagenesis, a subtractivecircular dichroism analysis was performed (see example 3). A comparisonof signals obtained from 250 to 184 nm for the wild type Z domain andthe four variants fused to the ABP-tail was performed after subtractionof the contribution from the ABP tail itself. The result showed that forthree of the four derivatives spectra similar to the wild type Z domainwere obtained, with a characteristic minimum at 208 nm and an inflexionpoint at 222 nm (Johnson, (1990) Prct. Struct. Funct. Genet., 7,205-224) (FIG. 17). This suggests that the three helix bundle frameworkprobably is preserved in these mutants. However, for the fourthderivative (no. 24), a spectrum was obtained which resembles spectraseen for random coils, indicating a low content of secondary structureelements (Johnson, 1990). This derivative contains a glutamine toproline substitution at position 32 in helix 2, suggesting adestabilization leading to a collapse of the helix bundle framework.

In order to further investigate the four Z-variants, the interactionwith polyclonal human IgG (hIgG) (Pharmacia AB) for wild type Z and fourdifferent Z variant clones (no. 16, 21, 22, 24; FIG. 14) fused to theABP tail were compared using biosensor technology (BIAcore™, PharmaciaBiosensor AB, Sweden). The carboxylated dextran layer of a CM-5 sensorchip was activated using N-hydroxysuccinimide (NH5) and N-ethyl-N'-3-diethylaminopropyl!-carbodiimide (EDC) chemistry according to themanufacturers' recommendations. For immobilization of hIgG, 20 μl of a500 nM hIgG solution in 50 mM acetate, pH 4 was injected at a flow rateof 5 μl/min over the activated surface, resulting in the immobilizationof approximately 5000 resonant units (RU). Fortyfive-microlitre samplesof the five fusion proteins, dissolved to approximate concentrations of1500 nM in NaCl/Hepes (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA,0.5% surfactant P-20), were injected in separate experiments at a flowrate of 2 μl/min. After each sample injection, the hIgG surface wasregenerated with 20 mM HCl. As expected, only the wild type Z-domainshowed any detectable Fc-binding activity (FIG. 18).

In conclusion, the results show that a library of SPA variants with asubstituted surface made up from 13 residues located in the α-helicescan be constructed. The high degree of conservation of the overallframework of the native Z-domain suggests that derivatives with novelfunctions grafted onto a stable and soluble scaffold could be isolatedfor use as artificial antibodies in biochemistry, immunology andbiotechnology.

We claim:
 1. A modified binding protein derived from a native (wild-type) bacterial receptor having an ααα helical domain structure, wherein said modified binding protein derived from said bacterial receptor comprises the B domain of staphylococcal protein A, and wherein said modified binding protein is produced by mutagenesis of amino acid residues 9, 11, 14, 27 and 35 of said B domain structure, which results in a modified binding protein which specifically binds to a different binding partner than the unmodified native (wild-type) bacterial receptor, and which retains the structure and stability of the native (wild-type) bacterial receptor.
 2. The modified binding protein of claim 1, wherein said modifications result in a glutamic acid residue at position 9, an aspartic acid at position 11, an aspartic acid at position 14, an alanine at position 27, and either a glutamic acid or alanine at position 35 of said B domain.
 3. A modified binding protein derived from a native (wild-type) bacterial receptor having an ααα 3-helix domain structure, wherein said modified binding protein derived from a bacterial receptor comprises the B domain of staphylococcal protein A, and wherein said modified binding protein is produced by mutagenesis of at least two of residues 9, 10, 11, 13, 14, 17, 28, 32, 35, 18, 24, 25 and 27 in the Z Scaffold of said B domain structure, and wherein said modifications result in a modified binding protein that specifically binds to a different binding partner than the unmodified native (wild-type) bacterial receptor, and which substantially retains the basic structure and stability of the native (wild-type) bacterial receptor. 